Apparatus for growing cells

ABSTRACT

A cell-culture apparatus forms an extra-capillary space between at least one hollow fiber and an enclosed chamber. Cells are placed in the extra-capillary space to grow. A media reservoir holds a cell-culture medium. The cell-culture medium is allowed to pass through a lumen of the at least one hollow fiber and to pass nutrients through the walls of the at least one hollow fiber to the cells in the extra-capillary space. Flow through the at least one hollow fiber is produced by action of gravity when a rocking or rotating motion is imparted to the media reservoir.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. application Ser. No. 09/885,920,filed Jun. 22, 2001, now U.S. Pat. No. 6,566,126.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention described herein relates to an apparatus and a method forgrowing suspension and adherent cells in vitro.

2. Background Art

Growing living cells in vitro is performed for a variety of purposes,including the production of cell derivatives, the preparation of viralvaccines, and the recovery of valuable cell by-products. Among thedevices that have been developed for growing cells in vitro, theshell-and-tube type arrangement has become fairly common, particularlyfor growing suspension and adherent cells.

These devices use semipermeable tube-shaped hollow fibers (i.e.,capillaries), contained within an outer shell, and configured so thatfluid within a space external to the hollow fibers (i.e., anextra-capillary space) is segregated from fluid passing through thehollow fibers and their corresponding openings (i.e., lumens).Additionally, these devices usually include two manifold end chamberswithin the outer shell on opposite ends of the device. Each of the twolumens of a hollow fiber connects to a different end chamber. The endchambers and the extra-capillary space are separated by thesemi-permeable membranes of the hollow fibers. The composition of theextra-capillary space can be controlled, to a certain extent, by themolecular weight cutoff, or pore size, of the membranes of the hollowfibers.

Typically, cells are grown in the extra-capillary space while a nutrientmedia is passed through the hollow fibers. The semipermeable nature ofthe hollow fibers allows nutrients and cell waste products to passthrough the walls of the hollow fibers while blocking cells from doingthe same. U.S. Pat. No. 4,391,912 to Yoshida et al. specifies a range ofpore diameters to support the transfer of the nutrient medium from theintra-capillary to the extra-capillary space while blocking the entranceof cells into the intra-capillary space.

Shell-and-tube type bioreactors provide several advantages. For adherentcells, the use of several hollow fibers provides, within a relativelysmall volume, a large amount of surface area upon which the cells cangrow. For both suspension and adherent cells, this large amount ofsurface area also facilitates localized distribution of nutrient mediato the growing cells and ready collection of cell waste products.Shell-and-tube type bioreactors enable the growth of cells at muchhigher density rates than is possible with other cell culture devices.They can support cell densities greater than 10⁸ cells per milliliter,whereas other cell culture devices are typically limited to densitiesaround 10⁶ cells per milliliter.

However, existing designs typically require external support systems tocirculate the nutrient media through the hollow fibers. U.S. Pat. No.3,883,393 to Knazek et al., U.S. Pat. No. 4,144,136 to Corbeil, U.S.Pat. No. 4,391,912 to Yoshida et al., U.S. Pat. No. 5,290,700 to Binotet al., and U.S. Pat. No. 5,955,353 to Amiot, all teach systems in whichthe nutrient medium is supplied to the intra-capillary spaces usingpumps and associated connection tubing. These external circulatingsystems add considerably to the cost of using these types ofshell-and-tube bioreactors.

What is needed is a shell-and-tube type apparatus to grow suspension andadherent cells that does not require an external circulating system forthe nutrient media fluid.

BRIEF SUMMARY OF THE INVENTION

The present invention describes an apparatus and method for growingcells. An extra-capillary space is formed between hollow fibers and anenclosed chamber. Cells are placed in the extra-capillary space to grow.A media reservoir holds cell-culture media. The cell-culture media isallowed to pass through a lumen of the hollow fibers and to passnutrients through the walls of the hollow fibers to the cells in theextra-capillary space. Flow through the hollow fibers is produced byaction of gravity when a rocking motion is imparted to the mediareservoir or by action of gravity when the media reservoir is rotatedabout a horizontal axis.

Preferably, the hollow fibers are made of a semi-permeable material. Thesemi-permeable material allows: (1) nutrients and (2) metabolizinggasses to pass from the cell-culture media through the walls of thehollow fibers to the cells in the extra-capillary space, and (3) cellwaste products and (4) gaseous waste products to pass from theextra-capillary space through the walls of the hollow fibers to thecell-culture media, while retaining the cells and large secretedproducts within the extra-capillary space.

Preferably, the media reservoir further includes a membrane permittinggas exchange between an exterior and an interior of the media reservoir.

Preferably, the media reservoir includes a first opening for accessingthe interior of the media reservoir. The first opening allows: (1) freshcell-culture media to be supplied to the media reservoir, (2) stalecell-culture media to be removed from the media reservoir, and (3) cellwaste products to be removed from the media reservoir.

In one embodiment, the enclosed chamber is disposed within the mediareservoir. An extra-chamber space is defined between the media reservoirand the enclosed chamber. Each hollow fiber has, at each end, a lumenopen to the extra-chamber space. Preferably, the media reservoirincludes a second opening for accessing the extra-capillary space. Thesecond opening allows: (1) developing cells to be placed into theextra-capillary space, (2) mature cells to be removed from theextra-capillary space, (3) secreted products to be harvested, and (4)the cells to be treated with reagents, drugs, and/or DNA or RNA vectors.

In another embodiment, the media reservoir is configured to cause a flowof the cell-culture media between a first port and a second port. Theenclosed chamber is connected between the first and second ports. Eachhollow fiber has, at each end, a lumen open to the flow of thecell-culture media between the first and second ports. Preferably, theenclosed chamber includes a second opening for accessing theextra-capillary space. The second opening allows: (1) developing cellsto be placed into the extra-capillary space, and (2) mature cells to beremoved from the extra-capillary space.

Further features and advantages of the invention as well as thestructure and operation of various embodiments of the present inventionare described in detail below with reference to the accompanyingdrawings.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

The accompanying drawings, which are incorporated herein and form partof the specification, illustrate the present invention and, togetherwith the description, further serve to explain the principles of theinvention and to enable a person skilled in the pertinent art to makeand use the invention.

FIG. 1 is a cutaway, cross sectional side view of a preferred embodimentof the apparatus of the present invention.

FIG. 2 is a cutaway end view showing a partial cross section of theembodiment shown in FIG. 1 taken along section A-A′.

FIG. 3 is similar to FIG. 1, but illustrates how a rocking motion causesthe flow of the cell-culture media.

FIG. 4 is a side view of an alternative preferred embodiment of theapparatus of the present invention.

FIG. 5 shows a top view of the embodiment shown in FIG. 4.

FIG. 6 is a flowchart illustrating the method 600 of the presentinvention.

The preferred embodiments of the invention are described with referenceto the figures where like reference numbers indicate identical orfunctionally similar elements. Also in the figures, the left most digitof each reference number identifies the figure in which the referencenumber is first used.

DETAILED DESCRIPTION OF THE INVENTION

FIG. 1 is a cutaway, cross sectional side view of a preferred embodimentof the apparatus of the present invention. In FIG. 1, a media reservoir102 holds cell-culture media 104 and is configured to be rocked orrotated about a horizontal axis of rotation 106 that extends into thedrawing sheet of FIG. 1. An enclosed chamber 108 is disposed withinmedia reservoir 102, wherein an extra-chamber space 110 is definedbetween media reservoir 102 and enclosed chamber 108.

A plurality of hollow fibers 112 pass through enclosed chamber 108 andare secured at each end by a first potting structure 114 and a secondpotting structure 116. An extra-capillary space 118 is defined betweenan interior of enclosed chamber 108 and the exterior surfaces of thehollow fibers 112. The hollow fibers 112 are oriented substantiallyparallel to a longitudinal axis 120, which is substantiallyperpendicular to horizontal axis of rotation 106.

In a representative embodiment of the invention, 25 to 200 hollow fibers112 are disposed within enclosed chamber 108. The length of the hollowfibers 112 is from about 3 to 10 centimeters, and the diameter is fromabout 200 to 1,000 microns. Preferably, hollow fibers 112 have alength-to-diameter ratio of less than about 170:1 to reduce the headloss of cell-culture media 104 that passes through them. Because thepresent invention relies on action of gravity to cause the flow ofcell-culture media 104, it is preferred that the hollow fibers 112 havea length-to-diameter ratio small enough to allow a sufficient rate offlow through them. Cumulatively, hollow fibers 112 can support acell-culture media flow rate from about 5 to 100 milliliters per minuteand have a surface area from about 25 to 1,000 square centimeters.However, one skilled in the art would recognize embodiments of thepresent invention with both a greater and a fewer number of hollowfibers 112 (including as few as one hollow fiber 112) and with differentparameters defining them.

FIG. 2 is a cutaway end view showing a partial cross section of theembodiment shown in FIG. 1 taken along section A-A′. In FIG. 2, eachhollow fiber 112 has, at each end, a lumen 202 open to extra-chamberspace 110 such that cell-culture media 104 can pass through the lumen202 of hollow fiber 112 and pass nutrients through the walls of hollowfiber 112 to nourish the cells in the extra-capillary space 118.

Preferably, each hollow fiber 112 is made of a semi-permeable material.The semi-permeable material allows: (1) nutrients and (2) metabolizinggasses to pass from the cell-culture media 104 through the walls of thehollow fibers 112 to the cells in the extra-capillary space 118, and (3)cell waste products and (4) gaseous waste products to pass fromextra-capillary space 118 through the walls of the hollow fibers 112 tocell-culture media 104, while retaining the cells and large secretedproducts within extra-capillary space 118. Consumption of nutrients andmetabolizing gasses by the cells establishes a gradient betweenextra-chamber space 110 and extra-capillary space 118. The gradientcauses nutrients and gasses to diffuse through the walls of the hollowfibers 112 and into extra-capillary space 118. Conversely, a build up ofcell waste products and gaseous waste products in the extra-capillaryspace 118 establishes a reverse gradient between extra-capillary space118 and extra-chamber space 110. This reverse gradient causes thesewaste products to diffuse through the walls of the hollow fibers 112into the lumens 202 and finally into extra-chamber space 110.

Preferably, in one embodiment, the semi-permeable material has poreswith diameters no larger than 0.2 microns. In various embodiments, thesemi-permeable material can be made of one or more of polysulfone,modified polysulfone, polyvinyledine fluoride, cellulose acetate,acrylic copolymer, and a cellulose derivative, wherein said cellulosederivative is one or more of a mixed ester of cellulose andcupra-ammonium rayon. However, one skilled in the art will recognizethat other materials can be used for the semi-permeable material.

Returning to FIG. 1, media reservoir 102 preferably includes an opening122 for accessing extra-chamber space 110. Opening 122 allows: (1) freshcell-culture media to be supplied to media reservoir 102, (2) stalecell-culture media to be removed from media reservoir 102, and (3) cellwaste products to be removed from media reservoir 102. A lid 124 is usedto seal opening 122.

Preferably, media reservoir 102 includes an opening 126 for accessingextra-capillary space 118. In an embodiment, opening 126 includes a port128 passing through extra-chamber space 110 to provide access toextra-capillary space 118. Opening 126 allows: (1) developing cells tobe placed into extra-capillary space 118, (2) mature cells to be removedfrom extra-capillary space 118, (3) secreted products to be harvested,and (4) the cells to be treated with reagents, drugs, and/or DNA or RNAvectors. In an embodiment, media reservoir 102 includes more than oneopening 126.

Preferably, media reservoir 102 further includes a gas permeablemembrane 130 permitting gas exchange between an environment exterior tomedia reservoir 102 and extra-chamber space 110. As discussed above,gasses are exchanged between extra-chamber space 110 and extra-capillaryspace 118 through the walls of hollow fibers 112. Membrane 130 permitsthe exchange of the waste gasses from extra-chamber space 110 with freshgasses from the environment exterior to media reservoir 102. Transversemembers 134 provide support and structural integrity to media reservoir102 along a face that includes membrane 130. In one embodiment, membrane130 is made of silicone. However, one skilled in the art will recognizethat other materials can be used for the membrane.

A dam 136 is disposed in media reservoir 102 to impede flow ofcell-culture media 104 in extra-chamber space 110 when media reservoir102 is rocked or rotated about horizontal axis of rotation 106. Dam 136also serves to encourage flow of cell-culture media 104 through thehollow fibers 112. In an embodiment in which enclosed chamber 108 spansthe width of media reservoir 102 along horizontal axis of rotation 106,enclosed chamber 108 and dam 136 are integrated.

FIG. 3 is similar to FIG. 1, but illustrates how a rocking or rotatingmotion causes the flow of cell-culture media. In FIG. 3, one skilled inthe art will recognize how dam 136, by impeding the flow of cell-culturemedia 104 in extra-chamber space 110, simultaneously increases thestatic head pressure of a raised portion 302 of cell-culture media 104and decreases the static head pressure of a lowered portion 304 ofcell-culture media 104 that would otherwise exist in the absence of dam134. Thus, by increasing the differential pressure across the hollowfibers 112, dam 136 serves to encourage flow of cell-culture media 104through the hollow fibers 112.

FIG. 4 is a side view of an alternative preferred embodiment of theapparatus of the present invention. FIG. 5 shows a top view of theembodiment shown in FIG. 4. In FIGS. 4 and 5, a media reservoir 402holds cell-culture media 104 and is configured to cause a flow ofcell-culture media 104 between a first port 502 and a second port 504 inresponse to a rocking or rotating motion imparted to media reservoir402. An enclosed chamber 404 is connected between first port 502 andsecond port 504. One skilled in the art will recognize that theseconnections can be realized by several means including, but not limitedto, a first tubing section 506 connected between first port 502 andenclosed chamber 404, and a second tubing section 508 connected betweensecond port 504 and enclosed chamber 404.

A plurality of hollow fibers 510 passes through enclosed chamber 404 andis secured at each end by a first potting structure 512 and a secondpotting structure 514. An extra-capillary space 516 is defined betweenan interior of the enclosed chamber 404 and the exterior surfaces of thehollow fibers 510. Each hollow fiber 510 has, at each end, a lumen opento the flow of cell-culture media 104 between first port 502 and secondport 504 such that cell-culture media 104 can pass through the lumen ofhollow fiber 510 and pass nutrients through the walls of hollow fiber510 to nourish the cells in extra-capillary space 516. Other functionsand parameters of the hollow fibers 510 are identical to those includedin the first preferred embodiment described above.

In a representative embodiment of the invention, 25 to 200 hollow fibers510 are disposed within enclosed chamber 404. The length of the hollowfibers 510 is from about 3 to 10 centimeters, and the diameter is fromabout 200 to 1,000 microns. Preferably, hollow fibers 510 have alength-to-diameter ratio of less than about 170:1 to reduce the headloss of cell-culture media 104 that passes through them. Because thepresent invention relies on action of gravity to cause the flow ofcell-culture media 104, it is preferred that the hollow fibers 510 havea length-to-diameter ratio small enough to allow a sufficient rate offlow through them. Cumulatively, hollow fibers 510 can support acell-culture media flow rate from about 5 to 100 milliliters per minuteand have a surface area from about 25 to 1,000 square centimeters.However, one skilled in the art would recognize embodiments of thepresent invention with both a greater and a fewer number of hollowfibers 510 (including as few as one hollow fiber 510) and with differentparameters defining them.

Enclosed chamber 404 can be a standard, commercially availableshell-and-tube type bioreactor designed for use with an externalcirculating system.

Preferably, media reservoir 402 comprises a tray 406, a first bag 408connected to first port 502, and a second bag 410 connected to secondport 504. This configuration serves to encourage flow of cell-culturemedia 104 through the hollow fibers 510 when a rocking or rotatingmotion is imparted to media reservoir 402. Advantageously, thisconfiguration allows for flexibility in the orientation of hollow fibers510 with respect to horizontal axis of rotation 106. However, oneskilled in the art will recognize other embodiments by which mediareservoir 402 can be realized. These include, but are not limited to, aconfiguration using one bag connected to both ports, wherein the bagincludes a damming or dividing mechanism to encourage flow ofcell-culture media 104 through enclosed chamber 404 by increasing thedifferential pressure between the two ports when the bag is rocked orrotated.

In an embodiment, first bag 408 and second bag 410 are made of a gaspermeable material such as silicone permitting gas exchange between theenvironment exterior to media reservoir 102 and an interior 414 of mediareservoir 402. In an alternate embodiment, only a portion of bags 408,410 are made from a gas permeable material. Similar to the processdiscussed above, gasses are exchanged between an interior 412 of mediareservoir 402 and extra-capillary space 516 through the walls of hollowfibers 510. The gas permeable material permits the exchange of the wastegasses from the interior 412 of media reservoir 402 with fresh gassesfrom the environment exterior to media reservoir 402.

Preferably, media reservoir 402 includes an opening 518 for accessinginterior 412 of media reservoir 402. As described above, opening 518allows: (1) fresh cell-culture media to be supplied to media reservoir402, (2) stale cell-culture media to be removed from media reservoir402, and (3) cell waste products to be removed from media reservoir 402.A lid 414 is used to seal opening 518. Where media reservoir 402comprises first bag 408 and second bag 410, each bag has its own opening518.

Preferably, enclosed chamber 404 includes an opening 520 for accessingextra-capillary space 516. As described above, opening 520 allows: (1)developing cells to be placed into extra-capillary space 516, (2) maturecells to be removed from extra-capillary space 516, (3) secretedproducts to be harvested, and (4) the cells to be treated with reagents,drugs, and/or DNA or RNA vectors. In an embodiment, enclosed chamber 404includes more than one opening 520.

In an embodiment, enclosed chamber 404 is attached to tray 406 by a clip416. However, one skilled in the art would recognize other means bywhich enclosed chamber 404 could be held in an appropriate position withrespect to media reservoir 402.

FIG. 6 is a flowchart illustrating the method 600 of the presentinvention. In FIG. 6, at a step 602, the media reservoir is accessed tosupply cell-culture media. Where cell-culture media has previously beensupplied to the media reservoir, one skilled in the art will recognizethat step 602 can be omitted. At a step 604, cells are placed into theextra-capillary space formed between the semipermeable hollow fibers andthe enclosed chamber. At a step 606, the media reservoir is rocked backand forth to cause a flow of the cell-culture media to pass through thelumens of the hollow fibers by action of gravity. Alternatively, at step606, the media reservoir can be rotated about a horizontal axis ofrotation to cause a flow of the cell-culture media to pass through thelumens of the hollow fibers by action of gravity. The cell-culture mediapass nutrients through the walls of the hollow fibers to nourish thecells in the extra-capillary space. In an embodiment, the hollow fibersare oriented substantially perpendicular to the horizontal axis ofrotation.

Optionally, at a step 608, the extra-capillary space is accessed toremove the cells. Optionally, at a step 610, the media reservoir isaccessed to remove the cell-culture media and any waste products.

Conclusion

While various embodiments of the present invention have been describedabove, it should be understood that they have been presented by way ofexample, and not limitation. It will be apparent to persons skilled inthe relevant art that various changes in form and detail can be madetherein without departing from the spirit and scope of the invention.Thus the present invention should not be limited by any of theabove-described exemplary embodiments, but should be defined only inaccordance with the following claims and their equivalents.

1. An apparatus for growing cells, comprising: a media reservoir (orholding cell-culture media and configured to be rocked or rotated abouta horizontal axis of rotation; an enclosed chamber disposed within saidmedia reservoir, wherein an extra-chamber space is defined between saidmedia reservoir and said enclosed chamber; and at least one hollow fiberpassing through said enclosed chamber, wherein each said at least onehollow fiber is substantially oriented upon a longitudinal axissubstantially perpendicular to said horizontal axis of rotation, whereineach said at least one hollow fiber has, at each end, a lumen open tosaid extra-chamber space, and wherein an extra-capillary space isdefined between an interior of said enclosed chamber and an exteriorsurface of said at least one hollow fiber.
 2. The apparatus of claim 1,further comprising: a first opening in said media reservoir foraccessing said extra-chamber space; and a second opening in said mediareservoir for accessing said extra-capillary space.
 3. The apparatus ofclaim 1, wherein said at least one hollow fiber comprises a plurality ofhollow fibers.
 4. The apparatus of claim 3, wherein each of saidplurality of hollow fibers is made of a semi-permeable material.
 5. Theapparatus of claim 4, wherein said semi-permeable material is one ormore of polysulfone, modified polysulfone, polyvinyledine fluoride,cellulose acetate, acrylic copolymer, and a cellulose derivative,wherein said cellulose derivative is one or more of a mixed ester ofcellulose and cupra-ammonium rayon.
 6. The apparatus of claim 5, whereinsaid semi-permeable material has pores with diameters no larger than 0.2μm.
 7. The apparatus of claim 1, wherein said media reservoir comprisesa membrane permitting gas exchange between an exterior of said mediareservoir and said extra-chamber space.
 8. The apparatus of claim 7,wherein said membrane is made of silicone.
 9. The apparatus of claim 1,further comprising: a dam disposed in said media reservoir to impedeflow of said media in said extra-chamber space when said media reservoiris rocked or rotated about said horizontal axis of rotation and toencourage flow of said media through said at least one hollow fiber.